Coordination compounds derived from the interaction of streptomycin and cobalt, nickel, copper, and calcium salts characterized by 13C NMR and spectroscopic studies. Structure and bonding properties of the streptidine fraction

The coordination compounds of streptomycin (St), Co2(St)Cl4.13H2O (2), Co2(St)(NO3)4.7H2O (3), Ni2(St)Cl4.14H2O (4), Ni2(St)(NO3)4.14H2O (5), Cu2(St)Cl4.6H2O (6), and Ca(St)Cl2.8H2O (7) have been synthesized by the reaction of streptomycin sulfate (1) with three equivalents of the corresponding inorganic salt. The compounds (2)-(7) were characterized by electronic spectroscopy (in the solid state and in solution) by conductivity measurements and by 13C NMR in solution. The reaction of streptomycin with CuCl2 in water hydrolyzed the molecule giving the copper complex of the streptidine fraction (Std), Cu(Std)Cl.H2O (8). This compound was characterized by the same techniques. Detailed x-ray diffraction and 13C NMR studies of streptidine sulfate (9) were carried out.     

L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide): a novel thromboxane-prostaglandin endoperoxide antagonist

The effects of L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide) have been studied on pulmonary and other smooth muscle preparations in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-641,933 produced significant shifts in the dose-response curves to the prostaglandin endoperoxide analogues, U-44069 (pA2 7.06) and U-46619 (pA2 7.14), and prostaglandin (PG) F2 alpha (pA2 6.33) had minimal activity against contractions induced by histamine (pA2 4.38), 5-hydroxytryptamine (pA2 4.63), and acetylcholine (pA2 4.56) and slightly enhanced relaxation induced by PGE2. When tested on the guinea-pig gall bladder strip in vitro, L-641,953 antagonized contractions induced by U-44069 (pA2 7.03) but was less active against those induced by PGF2 alpha (pA2 6.03), PGE1 (pA2 5.62), and histamine (pA2 4.84). When tested in vitro on the guinea-pig pulmonary artery, L-651-953 significantly antagonized contractions induced by U-44069 (pA2 7.04), U-46619 (pA2 7.14), and PGF2 alpha (pA2 7.16) but was less effective against contractions induced by histamine (pA2 4.19). Schild analysis indicated that L-641,953 was fully competitive against contractions of either the guinea-pig tracheal chain induced by U-46619 or the guinea-pig pulmonary artery induced by U-44069 and U-46619. When tested on human platelets in vitro L-641,953 inhibited aggregation induced by U-44069 (IC50 1.3 X 10(-6) M) but not ADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-linked glycan structures of human lactoferrin produced by transgenic rice

Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc(2) (3.4%), Man(2)FucGlcNAc(2) (2.1%), Man(3)FucGlcNAc(2) (2.5%), Man(3)FucXylGlcNAc(2) (42.5%), two isomers of Man(2)FucXylGlcNAc(2) (39.1%), Man(3)XylGlcNAc(2) (6.5%), and Man(2)XylGlcNAc(2) (3.9%).     

Inhibition by divalent cations and sulphydryl reagents of the passive Ca2+ transport in human red blood cells observed in the presence of vanadate

The uptake of 45Ca2+ by human red blood cells induced by vanadate was found to be inhibited by a number of divalent cations. The following order of potencies was determined (in parentheses, IC50 in mmol/l): Cu2+ (0.006), Zn2+ (0.014), Cd2+ (0.030), Co2+ (0.20), Ni2+ (0.25), Mn2+ (8.0), Ba2+ (9.0), Sr2+ (14.0). The effects of Cu2+, Zn2+ and Cd2+ were biphasic--over a critical concentration their inhibitory potencies decreased, and finally, were lost. Besides Ca2+, Sr2+, Ba2+ and Mn2+ were also taken up, but only Ca2+ and Sr2+ were capable of eliciting the Gárdos effect. Ni2+ was not taken up. Several HS reagents also inhibited 45Ca2+ uptake. The following order of potencies was determined (in parentheses, IC50 in mmol/l): mersalyl (0.0025), 5,5'-dithiobis(2,2'-dinitrobenzoic acid) (0.011), p-chloromercuric acid (0.042), N-ethylmaleimide (2.0). The effects of all HS reagents except N-ethylmaleimide were biphasic. The biphasicity of the actions of the indicated agents was caused by the opening of a new pathway for 45Ca2+ entry which is different from that observed in the presence of vanadate alone, and is inhibited by low concentrations of these agents. The modified form of the anion channel seems to be identical with the former pathway. The last one is mediated by a transport protein which has an ionic specificity similar to Ca2+ channels in excitable tissues, and contains an HS group which is essential for the transport function.     

Preparation of retinamides by use of retinoyl fluoride

Retinoyl fluoride (2) prepared from retinoic acid (1) by reaction with diethylaminosulfurtrifluoride is a stable crystalline compound not easily hydrolyzed by water. By reacting retinoyl fluoride with water-soluble amines in the presence of sodium bicarbonate, retinamide (4), N-retinoyl glycine (6), N-retinoyl DL-phenylalanine (7), alpha-N-retinoyl-L-lysine (11), N-retinoyl 4-aminophenol (4-hydroxyphenylretinamide) (8), and N-retinoyl-2-amino-2-deoxy-D-glucose (2-deoxy-D-glucose-2-retinamide) (9) have been prepared in good yields and characterized by UV absorption, 1H NMR, IR spectra, mass spectrometry, and elemental analysis.     

A membrane network of receptors and enzymes for adenine nucleotides and nucleosides

Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y(1), P2Y(2), P2Y(12), P2Y(13), P2X(2), A(1), A(2A) receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y(1), P2Y(12), P2Y(13), A(1), A(2A), and P2X(2) receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y(1)/P2Y(1), A(2A)/P2Y(1), A(2A)/P2Y(12)) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.     

Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-kappaB pathways

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.     

The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.     

P2 purinergic receptors of human eosinophils: characterization and coupling to oxygen radical production

Extracellular nucleotides elicit multiple responses in eosinophils but no information on expression of purinergic receptors in these cells is available so far. In the present study we show that human eosinophils express the following P2Y and P2X subtypes: P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), and P2X(1), P2X(4), P2X(7), whose stimulation results in intracellular Ca(2+) increase and production of large amounts of reactive oxygen intermediates. These events are stimulated or inhibited, respectively, by P2 receptor agonists or antagonists.     

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2 alpha formation from prostaglandin H2 by various rat glutathione S-transferase isozymes

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).     

Characterization of inhibitory effects of perfluorooctane sulfonate on human hepatic cytochrome P450 isoenzymes: focusing on CYP2A6

Perfluorooctane sulfonate (PFOS) is a chemically stable compound extensively used as oil and water repellent, surface active agents in our daily life. Accumulative research evidence gradually appears the toxicity of PFOS against mammals, but the whole figure remains to be elucidated. The present study was conducted to know the effects of PFOS on human hepatic drug metabolizing-type cytochrome P450 (CYP) isoenzymes such as CYP1A2 (7-ethoxyresorufin as a substrate), CYP2A6 (coumarin), CYP2B6 (7-ethoxy-4-trifluoromethylcoumarin), CYP2C8 (paclitaxel), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (bufuralol), CYP2E1 (chlorzoxazone) and CYP3A4 (testosterone) in human livers employing their typical substrates. Although all of the oxidation reactions tested were more or less inhibited by PFOS, diclofenac 4'-hydroxylation mediated mainly by CYP2C9 was most strongly inhibited (K(i) value of 40 nM), followed by paclitaxel 6α-hydroxylation mediated mainly by CYP2C8 (K(i) value of 4 μM). The substrate oxidation reactions catalyzed by CYP2A6, CYP2B6, CYP2C19 and CYP3A4 were moderately (K(i) values of 35 to 45 μM), and those by CYP1A2, CYP2D6 and CYP2E1 were weakly inhibited by PFOS (K(i) values of 190-300 μM). The inhibition by PFOS for coumarin 7-hydroxylation mainly catalyzed by human liver microsomal CYP2A6 as well as by the recombinant enzyme was found to be enhanced by the preincubation of PFOS with human liver microsomes and NADPH as compared to the case without preincubation. The inhibition of the human liver microsomal cumarin 7-hydroxylation was PFOS concentration-dependent, and exhibited pseudo-first-order kinetics with respect to preincubation time, yielding K(inact) and K(I) values of 0.06 min(-1) and 23 μM, respectively. These results suggest that the metabolism of medicines which are substrates for CYP2C9 may be altered by PFOS in human bodies, and that PFOS is a mechanism-based inhibitor of CYP2A6.     

Liquid chromatographic-mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2)) produced by cultured cells. Samples were separated on a C(18) column with water-acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF(1alpha), PGD(2), PGE(2), PGF(2alpha), and PGJ(2), respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10(-6) M) stimulated a marked increase in the production of 6-keto-PGF(1alpha), PGE(2), and PGF(2alpha) and a small increase of PGD(2) by ECs. 6-Keto-PGF(1alpha) was the major metabolite in these cells. The production of PGE(2) was threefold higher and PGD(2) was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.     

南海中华小尖柳珊瑚中嘌呤和嘧啶类化合物的研究

对采自海南三亚的中华小尖柳珊瑚Muricella flexuosa的化学成分进行研究,采用反复硅胶柱色谱法、Sephadex LH-20柱色谱法及重结晶等手段对化合物进行分离和纯化,通过理化性质及光谱分析并结合文献对照,鉴定得到11个嘌呤、嘧啶类化合物:咖啡碱(1),1,7-二甲基次黄嘌呤(2),1-甲基次黄嘌呤(3),7,9-二甲基-6-氮甲基嘌呤-8-酮(4),7-甲基腺嘌呤(5),1,7-二甲基嘌呤-6,8-二酮(6),尿嘧啶(7),胸腺嘧啶(8)2,’-脱氧尿嘧啶核苷(9)2,’-脱氧胸腺嘧啶核苷(10),3-乙基-2’-脱氧尿嘧啶核苷(11)。其中化合物2～61,1为首次从该属中分离得到,化合物4和11为新的天然产物。     

Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS

Prostaglandin (PG) D(2) ethanolamide (prostamide D(2)) was reduced to 9alpha,11beta-PGF(2) ethanolamide (9alpha,11beta-prostamide F(2)) by PGF synthase, which also catalyzes the reduction of PGH(2) and PGD(2) to PGF(2alpha) and 9alpha,11beta-PGF(2), respectively. These enzyme activities were measured by a new method, the liquid chromatographic-electrospray ionization-mass spectrometry (LC/ESI/MS) technique, which could simultaneously detect the substrate and all products. PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2) were separated on a TSKgel ODS 80Ts column, ionized by electrospray, and detected in the negative mode. Selected ion monitoring (SIM) of m/z 353 ([M-H](-)), 353 ([M-H](-)), 351 ([M-H](-)), 333 ([M-H-H(2)O](-)), 456 ([M+59](-)), and m/z 358 ([M-37](-)) was used for quantifying PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2), respectively. The detection limit for PGF(2alpha) and 9alpha,11beta-PGF(2) was 0.01pmol; that for PGH(2) and PGD(2), 0.1pmol; and that for prostamide D(2) and 9alpha,11beta-prostamide F(2), 0.5 and 0.03pmol, respectively. The LC/ESI/MS technique for measuring PGF synthase activity showed higher sensitivity than other methods. Using this method, we found that Bimatoprost, the ethyl amide analog of 17-phenyl-trinor PGF(2alpha) and an anti-glaucoma agent, inhibited all three reductase activities of PGF synthase when used at a low concentration. These results suggest that Bimatoprost also behaves as a potent PGF synthase inhibitor in addition to having prostamide-like activity.     

Synthesis, structural characterization and biological study of new organotin(IV), silver(I) and antimony(III) complexes with thioamides

An overview of our work on the synthesis and biological activity of a series of tin(IV), silver(I) and antimony(III) complexes with thioamides is reported. Organotin(IV) complexes of formulae (n-Bu)2Sn(MBZT)2 (1), Me2Sn(CMBZT)(2) (2), {(Ph3Sn)2(MNA) (Me2CO)} (3), Ph3Sn(MBZT) (4), Ph3Sn(MBZO) (5), Ph3Sn(CMBZT) (6), Ph2Sn(CMBZT)2 (7) and (n-Bu)2Sn(CMBZT)2 (8), Me2Sn(PMT)2 (9), (n-Bu)2Sn(PMT)2 (10), Ph2Sn(PMT)2 (11), Ph3Sn(PMT) (12) {where MBZT=2-mercapto-benzothiazole, CMBZT=5-chloro-2-mercapto-benzothiazole, H2MNA=2-mercapto-nicotinic acid, MBZO=2-mercapto-benzoxazole and PMTH=2-mercapto-pyrimidine} were characterized by spectroscopic (NMR, IR, Mossbauer, etc.) and X-ray diffraction techniques and their influence on the peroxidation of oleic acid was studied. They were found to inhibit strongly the peroxidation of linoleic acid by the enzyme lipoxygenase. In addition, organotin(IV) complexes were found to exhibit stronger cytotoxic activity in vitro, against leiomyosarcoma cells, than cisplatin. The antiproliferative activity of the organotin complexes studied, against leiomyosarcoma cells follow the same order of LOX activity inhibition. This is, 3>12>7>6 approximately 8 approximately 10>5 approximately 4>2>9. Thus, among organotin(IV)-CMBZT complexes, 7 exhibits higher activity than the others and this is explained by a free radical mechanism, as it is revealed by an EPR study. The results are compared with the corresponding ones found for the silver(I) complexes of formulae complexes {[Ag6(mu3-HMNA)4(mu3-MNA)2](2-).[(Et(3)NH)+]2.(DMSO)2.(H2O)} (13), {[Ag4Cl4(mu3-STHPMH2)4]n} (14), {[Ag6(mu2-Br)6(mu2-STHPMH2)4(mu3-STHPMH2)2]n} (15), {[Ag4(mu2STHPMH2)6](NO3)4}(n) (16), {[AgCl(TPTP)]4} (17), [AgX(TPTP)3] with X=Cl (18), Br (19), I (20) (where STHPMH2=2-mercapto-3,4,5,6-tetrahydro-pyrimidine, TPTP=tri(p-toly)phosphine) and those of antimony(III) complexes {[SbCl2(MBZIM)4](+).Cl(-).2H2O.(CH3OH)} (21), {[SbCl2(MBZIM)4]+.Cl(-).3H2O.(CH3CN)} (22), [SbCl3(MBZIM)2] (23), [SbCl3(EMBZIM)2] (24), [SbCl3(MTZD)2] (25), {[SbCl3(THPMT)2]} (26) and {[Sb(PMT)3].0.5(CH3OH)} (27) (where MBZIM is 2-mercapto-benzimidazole, EMBZIM=5-ethoxy-2-mercapto-benzimidazole and MTZD is 2-mercapto-thiazolidine), which they have characterized with similar techniques as in case of organotin(IV) complexes. Silver(I) and antimony(III) complexes were found to be cytotoxic against various cancer cell lines.     

Functional crosstalk between phospholipase D(2) and signaling phospholipase A(2)/cyclooxygenase-2-mediated prostaglandin biosynthetic pathways

We performed reconstitution analyses of functional interaction between phospholipase A(2) (PLA(2)) and phospholipase D (PLD) enzymes. Cotransfection of HEK293 cells with cytosolic (cPLA(2)) or type IIA secretory (sPLA(2)-IIA) PLA(2) and PLD(2), but not PLD(1), led to marked augmentation of stimulus-induced arachidonate release. Interleukin-1-stimulated arachidonate release was accompanied by prostaglandin E(2) production via cyclooxygenase-2, the expression of which was augmented by PLD(2). Conversely, activation of PLD(2), not PLD(1), was facilitated by cPLA(2) or sPLA(2)-IIA. Thus, our results revealed functional crosstalk between signaling PLA(2)s and PLD(2) in the regulation of various cellular responses in which these enzymes have been implicated.     

Inhibition of wound ethylene in Lycopersicum esculentum fruit discs by 1-amino cyclopropane-1-carboxylic acid oxidase inhibitors

Inhibition of wound-ethylene by eight structural analogues of 1-aminocyclopropane-1-carboxylic acid (ACC) studied seperately was investigated in unripe tomato fruit discs (Lycopersicum esculentum). The compounds tested were: trans-2-phenylcyclopropane-1-carboxylic acid (PCCA), cyclopropane-1,1-dicarboxylic acid (CDA), cyclopropylamine (CPA), cyclopropyl methyl ketone (CMK), chrysanthemyl alcohol (CHRA), 2-methyl cyclopropanecarboxylic acid (MCA), cyclopropanecarboxylic acid (CCA), 2-methyl-cyclopropane-methanol (2-MCM). The level of inhibition was a function of treatment concentration and time. Differential inhibition induced by the tested compounds was related to their structure.     

A novel receptor on allograft (H-2d)-induced macrophage (H-2b) toward an allogeneic major histocompatibility complex class I molecule, H-2Dd, in mice

The generation of knockout mice demonstrated that noncytotoxic CD4(+), but not cytotoxic CD8(+), T cells were essential for the rejection of skin or organ allografts. Earlier we reported that allograftinduced macrophages (AIM) in mice lysed allografts with H-2 haplotype specificity, implying screening of grafts by AIM. Here, we isolated a cDNA clone encoding a novel receptor on AIM (H-2D(b)) for an allogeneic major histocompatibility complex (MHC) class I molecule, H-2D(d), by using H-2D(d) tetramer and a monoclonal antibody (mAb; R15) specific for AIM. The cDNA (1,181-bp) encoded a 342-amino acid polypeptide with a calculated molecular mass of 45 kDa and was found to be expressed on AIM, but not on resident macrophages or other cells, infiltrating into the rejection site. HEK293T cells transfected with this cDNA reacted with R15 mAb and H-2D(d), but not H-2L(d), H-2K(d), H-2D(b), H-2K(b), H-2D(k), or H-2K(k), molecules; and the H-2D(d) binding was suppressed by the addition of R15 or anti-H-2D(d) mAb. AIM yielded a specific saturation isotherm in the presence of increasing concentrations of H-2D(d), but not H-2D(b) or H-2D(k), molecules. The dissociation constant of AIM toward H-2D(d) tetramers was 1.9 x 10(-9) M ; and the binding was completely inhibited by the addition of R15 or anti-H-2D(d) mAb. These results reveal that a novel receptor for an allogeneic H-2D(d) molecule was induced on effector macrophages responsible for allograft (H-2(d)) rejection in H-2(b) mice.     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).